A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds. Fluorophores are sometimes used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator (when its fluorescence is affected by environment such as polarity or ions). But more generally it is covalently bonded to a macromolecule, serving as a marker (or dye, or tag, or reporter) for bioactive reagents (antibodies, peptides, nucleic acids). Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, i.e., fluorescent imaging and spectroscopy.
Small organic dyes capable of exhibiting switch-on fluorescence through sequence specific interaction with nucleic acids play an important role in fluorescence spectroscopy, diagnostics, imaging and biomedical applications. Selective targeting of double-stranded (ds) DNA using organic dyes offers powerful strategies to develop i) probes for molecular biology and immunohistochemistry, flow cytometry and DNA quantification, ii) genome-specific binders of potential therapeutic interest, and iii) treating gene-related human diseases especially cancer, parasitic and viral infections. In this regard, various sequence-specific small fluorescent molecules have been developed for biological assays including cell imaging and DNA-quantification in cells.
The discovery of DNA as a genetic material and its double helical structure has led to numerous studies directed at understanding DNA-small molecules interaction. Typically, small molecules interact with DNA in two modes viz. intercalation and minor groove binding among other interactions. The research efforts of Dervan and Lown set forth the foundation for developing series of small molecules with affinity for binding to adenine-thymine (AT)-rich minor groove of B-DNA.
4′,6-Diamidino-2-phenylindole (DAPI) and bis-benzamides (Hoechst dyes) are some of the well-studied minor groove nuclear staining agents. Unfortunately, these blue emitting DNA binders require ultraviolet (UV) light for excitation and the prolonged UV-illumination is known to cause cellular DNA damage through free radical generation which eventually leads to cell death.
Despite the selective binding to AT-rich minor groove of DNA, under certain conditions, Hoechst dyes are prone to bind GC-rich sites and also show binding affinity towards single-stranded (ss) DNA and RNA. Similarly, DAPI can bind to RNA and high concentrations are required for imaging, limiting its use only to fixed cells. Recently, cyan and green fluorescent DNA-selective probes such as BENA435 and C61 have been reported. However, these probes upon binding to DNA show fluorescent enhancement with low quantum yields. Thomas et al. recently reported a dinuclear ruthenium(II) polypyridyl complex as a DNA-staining probe, but high concentration of dye is required for cellular imaging.
Apart from these molecules, a large number of cyanine dyes have been extensively used in DNA gel staining, microchip-based DNA sensing and fluorescence staining of DNA in cell. Among the cyanine-family dyes, thiazole orange (TO) and yellow orange (YO) are two important classes of fluorescence probes which show significant fluorescence enhancement upon binding with DNA. Further, the homodimeric forms of TOTO-1 and YOYO-1 are found to be highly sensitive for DNA detection. However, these cyanine-based probes shows significant fluorescence enhancement in presence of RNA and ssDNA. Later, two other class of cyanine dyes such as SYBR® green I and PicoGreen I have been developed and successfully used for DNA staining in picogram scale, although they show fluorescence enhancement in presence of ssDNA.
The limitations of existing probes discussed above necessitate the need for developing highly specific DNA-selective probes with: i) long-wavelength excitation/emission, ii) strong switch-on fluorescence, iii) increased cell permeability, iv) non-toxicity to live cells and v) base pair specificity and fidelity to double stranded (ds) DNA.
Further, Alzheimer's disease (AD) and Frontotemporal dementia are forms of dementia characterized by deposition of amyloid plaques in the brain. These plaques are majorly composed of peptides Aβ40 and Aβ42, which are derived from neural cell surface protein called amyloid precursor protein (APP) by the action of secretase enzymes (β and γ secretase).
There is no cure for AD, once diagnosed based on cognitive state, patient is already in an advanced stage which worsen as time progresses and finally leads to death. The only strategy which could help the patient is the early diagnosis of Alzheimer's disease (AD), which is a major concern in the present situation.
The neurodegeneration and subsequent progressive deterioration in cognitive ability are hallmark symptoms of this incurable syndrome. The Aβ42 peptide with 42 amino acids has been shown to be highly susceptible to aggregation and toxic behavior among all the Aβ peptides. Thus, Aβ42 is an attractive biomarker to target for diagnosis and therapeutics of AD. One of the major problems in the diagnosis of AD is the lack of effective methods for the early detection of Aβ42 aggregates. While diagnosis of AD is traditionally based on behavioral tests or cognition in patients, several imaging technologies such as positron emission tomography (PET),5 magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) have been developed for the detection of Aβ42 aggregates. However, these technologies are still limited by several obstacles, like long data acquisition time, radioactive exposure, poor resolution and need of expensive equipment. Optical imaging using fluorescent and colorimetric probes has emerged as a potential alternative technique as it offers real-time, non-radioactive, high-resolution imaging for inexpensive diagnostics and screening of drugs for AD.
Thioflavin T (ThT) is the most extensively used fluorescence probe for the in vitro detection and staining of Aβ aggregates. However, it suffers from poor selectivity and often leads to false detection. In the past few years, derivatives of oxazine, BODIPY, curcumin, styryl have been developed and used as fluorescence probes for Aβ aggregates.
An ideal fluorescence probe must exhibit certain characteristic properties to be used as a diagnostic probe for Aβ aggregates in AD viz., i) high specificity and strong binding affinity, ii) emission in the optical window of 500-750 nm with a large Stokes shift, iii) switch-on fluorescence change upon binding with Aβ aggregates, and iv) ability to rapidly cross the blood brain barrier (BBB). Further, mixed dementia is a condition in which abnormal characteristics of more than one type of dementia occur simultaneously and, in such cases, determining the specific type of neurodegenerative disorder in the patient is very crucial. Therefore, there is an urgent need for developing probes which could selectively differentiate toxic aggregates. Unfortunately, there is lack of studies on probes that selectively differentiate plaques responsible for any specific disorder. Even in fluorescence probes, selectivity is the major issue, as most of them fluoresce upon binding to forced or artificially formed protein aggregates generally observed in all kinds of dementia. Moreover, colorimetric detection of Aβ aggregates using antibodies has been demonstrated; however, this technique is complicated and expensive.
ThT has been extensively used to stain Aβ aggregates for the past few decades. This probe mainly consists of electron donating (N,N-dimethylaniline) and electron withdrawing (benzothiazole) moieties. However, as mentioned above, this probe suffers from many drawbacks.
Therefore, there is a need for developing selective fluorometric and colorimetric probes based on simple organic molecules, which are easy to handle and offer quick detection and overcome the drawbacks of the prior art dyes/probes and function as selective probes for Aβ aggregates compared to other protein aggregates.
The present disclosure overcomes the limitations of the prior art by providing small molecular probes which are highly specific to AT-rich sequences of DNA vis-à-vis single stranded DNA, RNA and monomeric proteins and probes specific for Aβ aggregates compared to other protein aggregates.